| Eriobotrya japonica,is a plant of the genus Eriobotrya in the family Rosaceae and one of the major fruit trees in the south of China,which has a long history of cultivation and has been popular since ancient times.Loquat fruit ripens in late spring and early summer and is typically one of the most important off-season fruits.Among more than 20 species of loquat plants,only one species of loquat is widely cultivated and used in fruit tree production.Therefore,the genetic background of loquat is very narrow,which is very unfavorable for the improvement of more traits of loquat.China is the native country of loquat,and loquat genus is also abundant in the wild,and some of the wild has excellent traits(drought resistance,cold resistance,insect resistance,low seed number,etc.),so it is important to transfer some of the excellent traits of wild loquat into cultivated loquat for loquat production.The flowering of E.cavaleriei in early summer(April-May)and fruit ripening in September-November,and the absence of obvious diseases in the field and in mixed plantings with loquat,may be important for changing the flowering and ripening period and disease resistance of loquat.In the present study,the loquat cultivars ’Jinhua 2’,’Huabai 2’,’Xingning 1’ and tetraploid(2n=2x=34)with E.cavaleriei(2n=2x=34)as the parent and loquat cultivars and tetraploid(2n=4x=68)strains B433,B456,and K474 as the parent were analyzed to screen true hybrids and then analyze their karyotypes,leaf morphology,flesh color genotypes,and resistance to anthracnose to provide important materials for loquat breeding at different flowering and ripening stages,and also to provide reference for loquat heterozygous polyploid breeding.The main research contents are as follows:1、Leaf morphology observationThe leaves of Eriobotrya cavaleriei were oblanceolate,bright,with eccentric tips and flat margins.The morphological characteristics of the leaves of the hybrid progeny were observed and determined.The leaf shape of the hybrid offspring was mostly oval like that of the maternal parent,and the leaf margin shape was mostly flat like that of the paternal parent,with the leaf tip shape moving toward the middle.2、Hybrid identification using InDel molecular markersSixty-five pairs of InDel primers were used to amplify the DNA from the parents of the crosses.Seven pairs of primers with clear bands and good repeatability were selected by non-denaturing polyacrylamide gel electrophoresis.Four pairs of different primers were selected from the combination of ’Xingning 1’,’Huabai 2’ and B433 with Eriobotrya cavaleriei.Three pairs of different primers were selected from the hybridization combination of ’Jinhua 2’,K474 and B456 with Eriobotrya cavaleriei.The 7 pairs of primers were used to identify 100 loquat hybrids,and 93 true hybrids were identified,with a hybridization success rate of 93%.3 、 Detection of ploidy and determination of karyotype analysis of hybrid progenyFlow cytometry was used to identify the ploidy of 100 hybrid progenies,and 13 triploid materials were obtained.The ploidy of polyploid materials was verified by simplified genome re-sequencing.Four triploid true hybrids B433×Ec-3,B433×Ec-4,B433×Ec-5 and K474×Ec-5 were aneuploid.in which based on the sequencing depth the copy number of chromosome 13 of B433×Ec-3 and B433×Ec-5 and the copy number of chromosome 15 of K474×Ec-5 were 2,the copy number of chromosome 3 and 4 of B433×Ec-4 was 2 and the copy number of chromosome 6was 4.In addition,the proportion of the parental genome in the triploid hybrid genome was determined by drawing the scatter plot of the allele frequency of the hybrid using the simplified genome resequencing results(the proportion of cultivated loquat genome was 60%-70%,and that of wild loquat genome was 30%-40%).4、Pulp color genotyping of hybrid polyploid offspringAccording to the polyploid true hybrids(13 triploids)obtained in the hybrid identification,q PCR technology was used to identify the pulp color of the polyploid true hybrids.The results showed that there were 7 red and white meat heterozygous materials with genotype AAa and 6 red meat homozygous materials with genotype AAA in the triploid materials.Because the pulp color genotype of the paternal loquat was AA and red meat homozygous,the white meat homozygous loquat(genotype aaa or aaaa)could not be screened in the hybrid offspring.5、Preliminary analysis of anthracnose resistance in hybrid progenyColletotrichum gloeosporioides was used as the pathogen to test the resistance of the progeny of interspecific crosses to Colletotrichum gloeosporioides.The results showed that 62 accessions of the 74 hybrids were moderately resistant,8 accessions were highly resistant and 4 accessions were susceptible to the disease.All accessions were resistant to the disease beyond the resistance level of the parent,indicating that the hybridization between wild loquat and cultivated loquat could improve the resistance to a certain extent.In summary,this study was conducted to identify the authenticity of hybrids by observing the morphological characteristics of the leaf progeny,using InDel molecular markers and flow cytometry;triplets with parental characteristics were obtained,which provided important materials for subsequent heterologous polyploid breeding of loquat;aneuploidy was clearly identified in the progeny of tetraploiddiploid crosses,and all chromosomes with copy number changes were from loquat tetraploids,providing a reference for the creation of loquat allopolyploids;genotyping the flesh color of the hybrid polyploid progeny and preliminary evaluation of anthrax resistance in the hybrid progeny,clarifying the flesh color genotype of triploid material in the hybrid progeny,laying a certain foundation for the subsequent selection and breeding of loquat varieties with characteristic white flesh and anthrax resistance. |
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